1. Field of the Invention
This invention relates generally to increasing cytosolic Ca2+ levels in mammalian cells by triggering Ca2+ entry from extracellular “stores” (e.g., by Ca2+ from the solution vehicle entering the cell).
The invention has broad applicability in the pharmaceutical industry in screening for agents that stimulate Ca2+ entry in methods of treating bacterial infections, inflammatory conditions and diseases, airway diseases and infections (such as cystic fibrosis, asthma, common cold, lung and airways bacterial infections), gastrointestinal (GI) diseases and infections (cystic fibrosis, GI bacterial infections), kidney diseases (like polycystic kidney disease, salt-sensitive hypertension, and other renal hypertension syndromes), and endocrine and neuroendocrine disorders such as type I and type II diabetes, Alzheimer's disease, Addison's disease, pituitary dwarfism (pituitary GH secretion insufficiency), and amylotrophic lateral sclerosis (ALS or Lou Gehrig's disease).
2. Background Art
The epithelial lining of the respiratory tract contains both mucous-secreting goblet cells and ciliated cells. These cells are bathed in fluid and a layer of mucous rests on top of the cilia. In healthy lungs, the ciliated cells beat the mucous layer that contains inhaled foreign particles and microorganisms toward the larger bronchi and trachea, where it is either expectorated or swallowed. (Davies, Z. A. NurseWeek.com, May 7, 2002). In airway diseases, these functions are impaired.
In subjects with cystic fibrosis (CF), for example, the fluid and the mucous layer are dehydrated. The dehydrated mucous is thick and sticky, which inhibits the cilia from propelling mucous out of the airway. It accumulates and progressively obstructs the bronchioles and bronchi. The sticky mucous also creates an ideal environment for the proliferation of bacteria.
In CF, cyclic AMP- and protein kinase A-dependent transepithelial Cl− transport is impaired because of mutations in the CF gene that encodes for the protein, the cystic fibrosis transmembrane conductance regulator, or CFTR (Riordan et al, Science 245:1066-1073, 1989). Originally, CFTR was thought to function exclusively as a low conductance Cl− channel (Gregory et al, Nature 27:382-386, 1990; Berger et al, J. Clin. Invest. 88:1422-1431, 1991). More recently, it has become clear that CFTR also regulates a series of other transporters and ion channels, such as the Cl−/HCO3 exchanger, the NaHCO3 cotransporter, epithelial Na+ channels (ENaCs), K+ channels, and aquaporin water channels (Schreiber et al., Pflugers Arch. 434:841-847, 1997). In particular, ENaCs, a protein separate from but regulated by CFTR, are upregulated or hyperactive in CF are another class of dysregulated ion channel that needs correction with a CF therapeutic. Impaired Cl− transport is shared as a key disease phenotype by CF epithelia from all affected tissues and that this pathway is lost in CF. Therefore, activation of a cAMP-independent Cl− secretory pathway through exploitation of a naturally expressed epithelial protein is of interest for therapy in CF and other airway diseases.
Increases in Ca2+ activate epithelial chloride (Cl−) and potassium (K+) channels (Rugolo et al., J. Biol. Chem. 268:24779-24784, 1993; Tarran et al., J. Gen. Physiol. 120:407-418, 2002); however, epithelial Na+ channels (ENaCs) are inhibited by Ca2+ increases in epithelia. Thus, needed in the art are methods and a composition for selectively stimulating Ca2+-activated Cl− channels (CaCCs) as a possible substitute for cAMP-dependent cystic-fibrosis transmembrane conductance regulator (CFTR) Cl− channels impaired in cystic fibrosis (Fuller C M, Benos D J. News Physiol. Sci. 15: 165-171, 2000).
A subset of CF patients also have GI abnormalities, mostly related to pancreatic insufficiency, a loss of secretion of pancreatic enzymes and bicarbonate, and inadequate digestion and absorption of essential nutrients. Although malabsorption of elements like zinc has been reported in subjects with CF (Krebs et al. Pediatr. Res. 48: 256-261, 2000), such elements have not been used to treat airway diseases. Zinc is a trace element that is derived from diet and is enriched in certain tissues (Troung-Tran et al. Immunol. Cell Biol. 79: 170-177, 2001). Zinc oxide creams alleviate dermatitis, including acrodermatits enteropathica in CF patients caused by zinc malabsorption and deficiency. Defective activity of a zinc transporter is linked to this form of dermatitis (Wang et al. Am. J. Human Genet. 7: 66-73, 2002). Zinc also alleviates symptoms of or speeds recovery from rhinoviral infections. Homeopathic remedies such as Zicam™ and ColdEeze™, based on zincum gluconicum and administered either orally or as a nasal gel, are available over-the-counter to reduce symptoms and speed recovery from the common cold. Zinc has not been used previously to modulate Ca2+ entry.